The detection of antibodies by indirect immunofluorescence (IIF) employing different cellular and tissue substrates as multiple antigen sources has been established in routine diagnostics. Cell-based immunofluorescence tests, such as the detection of anti-nuclear antibodies (ANA) using HEp-2-cells, are widely used in clinical diagnostics and research as the industry standard (Tan E M, Adv. Immunology 1982; 33:167-240, Costes S V, et al., Radiat Res. 2006; 165(5):505-15, Sack U, et al., Ann N Y Acad Sci 2009; 1173:166-73, Conrad K, et al., Ann N Y Acad Sci 2009; 1173:180-185). Methods for diagnosing autoimmune diseases that use indirect immunofluorescence-based approaches are known in the art. WO 97/06440 discloses a method whereby rheumatoid arthritis is diagnosed by the presence of antibodies that are directed against microtubule organising centres, or microtubules extending therefrom, in a body fluid sample of a patient. The cell line used as substrate is preferably an IT-1 macrophage cell line, which is used as a substrate to which autoantibodies present in the patient sample bind.
The increasing demand for automated reading and interpretation of fluorescence patterns, resulting in improved standardization and cost-effectiveness, has been enabled by sophisticated pattern recognition software and fully automated reading machines that are available at the present time (Hou Y N, et al., Radiat Res. 2009; 171(3):360-7, Hiemann R, et al., Ann N Y Acad Sci 2007; 1109:358-71, Böcker W, et al., Radiat Res. 2006; 165(1):113-24. Hiemann R, et al., Autoimmun Rev. 2009; 9:17-22). Indirect immunofluorescence has additionally been applied in quantitative and semi-quantitative approaches to determining antibody titre, for example in the diagnosis of diseases characterised by the presence and/or quantity of autoantibodies (WO 2009/062479 A2).
However, advances in assay development and recombinant technology have paved the way for the detection of autoantibody specificities to individual antigenic targets thereby improving the diagnostic power of antibody testing. The growing variety of antibodies found in different disorders such as infectious and rheumatic diseases has generated the need for innovative techniques, which overcome the drawbacks of single antibody detection and decrease cost and time in reporting results. Hence, multiplexed platforms have been developed recently to meet the demand of simultaneous determination of several antibody specificities in one sample. Multiplex assays based on fluorescent bead-based flow cytometry and microarray systems have proven to be powerful tools supporting higher throughput analysis and more comprehensive testing of patient samples (Avaniss-Aghajani E, et al., Clin Vaccine Immunol. 2007; 14:505-9, Lal G, et al., J Immunol Methods 2005; 296:135-47). Computer-assisted pattern recognition has also been used to analyse antigen arrays, so that antibodies that bind to a panel of disease associated antigens can be detected and correlated with a particular disease diagnosis (Binder S R et al, Clinical and Diagnostic Laboratory Immunology, December 2005, 1353-1357). Furthermore for the assessment of rheumatic-disease-specific antibodies. multiplex detection by immobilized fluorescence-coded microbeads using multicolor fluorescence can be used. This multicolor fluorescence analyses with pattern detection algorithms provide a common platform technique for the screening of ANA (Groβmann K, et al., Cytometry A. 2011 February; 79(2):118-25).
As an example, disease-specific autoantibodies (AAB) are a serological phenomenon of systemic rheumatic conditions and autoimmune liver disorders. In particular, the detection of ANA by IIF was one of the first techniques available in routine laboratories for the serological diagnosis of systemic rheumatic diseases. Despite the development of enzyme-linked immunosorbent assay (ELISA) and multiplexing technologies for the detection of disease-specific AAB, the screening for ANA by IIF assays still remains the standard method in the current multistage diagnostic approach. Recombinant or purified antigens can be provided on beads, which are subsequently analysed for antibodies that bind specifically to the selected antigens using enzyme immunoassays. Such approaches exhibit however significant disadvantages, due to severely differing sensitivities and specificities between different antigens (Hayashi N, et al, 2001, Clinical Chemistry, 47:9 1649-1659). Multiplex bead-based fluorescent assays have been published that show reasonable correlation with cell-based autoantibody diagnostic methods (Smith J, et al, 2005, Ann. N.Y. Acad. Sci. 1050: 286-294), although the low sensitivity for identifying IIF-positive control samples indicates that IIF approaches are still the favoured method (Nifli A P, et al, Journal of Immunological methods 311 (2006), 189-197).
Several substrates have been proposed for ANA IIF assays, however the screening for non-organ specific AAB on HEp-2 cells is the most established method. In general, assessment of ANA is followed by detection of specific AAB to for example extractable nuclear (ENA) and cytoplasmic antigens by immunoassays employing purified native or recombinant antigens. This two-stage approach exhibits the following benefits: (i) highly sensitive screening of the most frequent, clinically relevant non-organ specific AAB, (ii) optimal combination with other assay techniques for the down-stream differentiation of AAB reactivities based on the IIF pattern detected and the diagnosis suspected (e.g., SS-A/Ro and SS-B/La), (iii) assessment of clinically relevant AAB without the need for further testing (e.g., anti-centromere AAB), and (iv) evaluation of AAB only detectable by IIF in case of unknown autoantigenic targets or unavailable commercial assays.
Another example is the detection of anti-neutrophil cytoplasmic antibodies (ANCA) for the differential diagnosis of systemic vasculitides (Bosch X, et al., Lancet 2006; 368:404-18). Due to the occurrence of ANCA in these systemic autoimmune disorders the term ANCA-associated vasculitis (AASV) has been coined for these clinical entities. ANCA were discovered by IIF, which is still the recommended method to detect these antibody reactivities. ANCA exhibit typically two different staining patterns of fixed granulocytes in IIF; a speckled cytoplasmic pattern (cANCA) and a perinuclear pattern (pANCA). cANCA, frequently found in patients with Wegener's granulomatosis (WG), are directed primarily against proteinase 3 (PR3), in addition to other targets, while pANCA, occurring mainly in microscopic polyangiitis (MPA), are directed primarily against myeloperoxidase (MPO) (Van der Woude F J, et al., Lancet 1985; 1:425-9; Csernok E, et al., Nat Clin Pract Rheumatol. 2006 April; 2(4):174-5), in addition to other targets.
Estimations of antibody concentrations have been proposed to be helpful in the diagnosis and management of these clinical entities. Antibody values are usually associated with the severity of disease (Cohen Tervaert J W, et al., Arch Intern Med 1989; 149: 2461-5). However, there have been other target antigens for ANCA described in IIF that can be found in patients with non-AASV (Savige J, et al, Best. Pract. Res. Clin. Rheumatol. 19: 263-76, Savige J, et al., Am J Clin Path 2003; 120:312-8). Consequently, in accordance with recently established consensus guidelines a different technique in addition to IIF for the detection of ANCA such as ELISA is recommended.
However, the techniques employed for determining several antibodies by the two- or multi-stage approach mentioned above involve a plurality of constituent tests which are individually labelled. Regarding cost-effective serological diagnostics there is a clear demand to combine the detection of antibodies to cellular and tissue antigenic targets on the one hand, and to the purified and characterised proteins thereof on the other hand, into a single method with one label.
Contemporary protein characterisation by microarray technology does not alone provide a satisfactory solution. As the number of antigenic targets to be tested on a single microarray increases, the demand for associated manufacturing equipment, miniaturization and specialized materials and handling will render the production of such microarrays increasingly complex and cost-intensive. Other techniques, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), chromogenic assays, high-performance liquid chromatography (HPLC), gas chromatography-mass spectroscopy (GC-MS), and thin layer chromatography (TLC), exhibit the disadvantage of being limited in the number of analytes in antigenic form that can be assessed simultaneously. They are also time-consuming and require expensive equipment. In contrast, employing a HEp-2 cell substrate for ANA detection provides more than 1200 antigenic targets for antibody identification.